Linking Stochastic Dynamics to Population Distribution: An Analytical Framework of Gene Expression

We present an analytical framework describing the steady-state distribution of protein concentration in live cells, considering that protein production occurs in random bursts with an exponentially distributed number of molecules. We extend this framework for cases of transcription autoregulation and noise propagation in a simple genetic network. This model allows for the extraction of kinetic parameters of gene expression from steady-state distributions of protein concentration in a cell population, which are available from single cell data obtained by flow cytometry or fluorescence microscopy

High-Resolution Near-Field Raman Microscopy of Single-Walled Carbon Nanotubes

We present near-field Raman spectroscopy and imaging of single isolated single-walled carbon nanotubes with a spatial resolution of ≈25  nm. The near-field origin of the image contrast is confirmed by the measured dependence of the Raman scattering signal on tip-sample distance and the unique polarization properties. The method is used to study local variations in the Raman spectrum along a single single-walled carbon nanotube

Generalized Haldane Equation and Fluctuation Theorem in the Steady State Cycle Kinetics of Single Enzymes

Enyzme kinetics are cyclic. We study a Markov renewal process model of single-enzyme turnover in nonequilibrium steady-state (NESS) with sustained concentrations for substrates and products. We show that the forward and backward cycle times have idential non-exponential distributions: \QQ_+(t)=\QQ_-(t). This equation generalizes the Haldane relation in reversible enzyme kinetics. In terms of the probabilities for the forward ($p_+$) and backward ($p_-$) cycles, $k_BT\ln(p_+/p_-)$ is shown to be the chemical driving force of the NESS, $\Delta\mu$. More interestingly, the moment generating function of the stochastic number of substrate cycle $\nu(t)$, follows the fluctuation theorem in the form of Kurchan-Lebowitz-Spohn-type symmetry. When $\lambda$ = $\Delta\mu/k_BT$, we obtain the Jarzynski-Hatano-Sasa-type equality: $\equiv$ 1 for all $t$, where $\nu\Delta\mu$ is the fluctuating chemical work done for sustaining the NESS. This theory suggests possible methods to experimentally determine the nonequilibrium driving force {\it in situ} from turnover data via single-molecule enzymology.Comment: 4 pages, 3 figure

Fiber optical parametric oscillator for coherent anti-Stokes Raman scattering microscopy

We present a synchronously pumped fiber optical parametric oscillator for coherent anti-Stokes Raman scattering microscopy. Pulses from a 1 μm Yb-doped fiber laser are amplified and frequency converted to 779–808 nm through normal dispersion four-wave mixing in a photonic crystal fiber. The idler frequency is resonant in the oscillator cavity, and we find that bandpass filtering the feedback is essential for a stable, narrow-bandwidth output. Experimental results agree quite well with numerical simulations of the device. Transform-limited 2 ps pulses with energy up to 4 nJ can be generated at the signal wavelength. The average power is 180 mW, and the relative-intensity noise is much lower than that of a similar parametric amplifier. High-quality coherent Raman images of mouse tissues recorded with this source are presented

Single Cell Transcriptome Amplification with MALBAC

Recently, Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) has been developed for whole genome amplification of an individual cell, relying on quasilinear instead of exponential amplification to achieve high coverage. Here we adapt MALBAC for single-cell transcriptome amplification, which gives consistently high detection efficiency, accuracy and reproducibility. With this newly developed technique, we successfully amplified and sequenced single cells from 3 germ layers from mouse embryos in the early gastrulation stage, and examined the epithelial-mesenchymal transition (EMT) program among cells in the mesoderm layer on a single-cell level.Chemistry and Chemical Biolog

Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients

Circulating tumor cells (CTCs) enter peripheral blood from primary tumors and seed metastases. The genome sequencing of CTCs could offer noninvasive prognosis or even diagnosis, but has been hampered by low single-cell genome coverage of scarce CTCs. Here, we report the use of the recently developed multiple annealing and looping-based amplification cycles for whole-genome amplification of single CTCs from lung cancer patients. We observed characteristic cancer-associated single-nucleotide variations and insertions/deletions in exomes of CTCs. These mutations provided information needed for individualized therapy, such as drug resistance and phenotypic transition, but were heterogeneous from cell to cell. In contrast, every CTC from an individual patient, regardless of the cancer subtypes, exhibited reproducible copy number variation (CNV) patterns, similar to those of the metastatic tumor of the same patient. Interestingly, different patients with the same lung cancer adenocarcinoma (ADC) shared similar CNV patterns in their CTCs. Even more interestingly, patients of small-cell lung cancer have CNV patterns distinctly different from those of ADC patients. Our finding suggests that CNVs at certain genomic loci are selected for the metastasis of cancer. The reproducibility of cancer-specific CNVs offers potential for CTC-based cancer diagnostics.Chemistry and Chemical Biolog

Imaging Chromophores With Undetectable Fluorescence by Stimulated Emission Microscopy

Fluorescence, that is, spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging. However, many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay. Yet the detection of their absorption is difficult under a microscope. Here we use stimulated emission, which competes effectively with the nonradiative decay, to make the chromophores detectable, and report a new contrast mechanism for optical microscopy. In a pump-probe experiment, on photoexcitation by a pump pulse, the sample is stimulated down to the ground state by a time-delayed probe pulse, the intensity of which is concurrently increased. We extract the miniscule intensity increase with shot-noise-limited sensitivity by using a lock-in amplifier and intensity modulation of the pump beam at a high megahertz frequency. The signal is generated only at the laser foci owing to the nonlinear dependence on the input intensities, providing intrinsic three-dimensional optical sectioning capability. In contrast, conventional one-beam absorption measurement exhibits low sensitivity, lack of three-dimensional sectioning capability, and complication by linear scattering of heterogeneous samples. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distributions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of haemoglobin. For all these applications, sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, permitting nonfluorescent reporters for molecular imaging.Chemistry and Chemical Biolog